Project 1 will use in vitro and in vivo models to investigate signal transduction pathways that transduce activation of mixed lineage kinase type 3 (MLKS), because we hypothesize that pathologic activation of MLKS leads to neuroinflammation and loss of synaptic plasticity in HAND. Using 2nd generation small molecule MLK3 inhibitors (designated as our lead compound URMC-099 or their descendant congeners, URMC-099c*) developed by our industry partner, Califia Bio Inc in Project 2, we have demonstrated that URMC-099 blocks pro-inflammatory cytokine production from HIV-1 Tat-stimulated monocyte-derived macrophage and microglia in vitro, as well as prevents leukocyte trafficking in CNS vasculature and damage to synaptic architecture following stereotactic injection of Tat in the CNS in vivo. Additionally, we have demonstrated that MLKS inhibition with these inhibitors increases the neuronal Tri<B receptor for brain derived neurotrophic growth factor (BDNF), suggesting that MLKS inhibition may be important for trophic support of neuronal networks affected in HAND. Thus, we propose three specific aims to further our understanding of MLKS activation in HAND: (1) To assess the molecular mechanism(s) by which MLKS regulates leukocyte infiltration in the CNS; (2) To determine whether BDNF-mediated signaling events contribute to the neuroprotective efficacy of MLKS blockade in models for HAND; and (3) To assess the effect of MLKS blockade in a small animal model for HAND, using real-time in vivo imaging methods to measure leukocyte infiltration, microglial activation and synaptic engulfment. In all aims, use ofthe MLKS inhibitors URMC-099c* will help guide refinement of structure activity relationship (SAR) work conducted with our industry partner, Califia Bio, Inc in Project 2 as part of the testing funnel so that the URMC-099c* with the best index of neuroprotection, the best profile of pharmacokinetic (i.e. CNS-penetrant) characteristics, the most metabolically stable, and without systemic or CNS toxicity, can be tested in rodent and monkey models of HAND in Project 3. Using this iterative, interactive progression of studies between Projects 1, 2 and 3, at the conclusion of this project period, we expect to bring fon/vard the most promising URMC-099c* as our candidate for IND filing with the FDA.